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Glutathione peroxidase 4: A new player in neurodegeneration? 1
Bárbara R. Cardoso1*, Dominic J. Hare1,2, Ashley I. Bush1, Blaine R. Roberts1* 2
3
Running title: Glutathione peroxidase 4 role in neurodegeneration 4
1 The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, 5
Parkville, Victoria, Australia 6
2 Elemental Bio-imaging Facility, University of Technology Sydney, Broadway, New 7
South Wales, Australia 8
* Corresponding authors: 9
Bárbara R. Cardoso, The Florey Institute of Neuroscience and Mental Health, Kenneth 10
Myer Building, 30 Royal Parade, Parkville, Victoria, 3052 Australia; Ph: +61 450 11
633537; Email: [email protected] 12
Blaine R. Roberts, The Florey Institute of Neuroscience and Mental Health, Kenneth 13
Myer Building, 30 Royal Parade, Parkville, Victoria, 3052 Australia; Ph: +61 14
490356635; Email: [email protected] 15
16
17
18
Abstract 19
The selenoprotein glutathione peroxidase 4 has been recognized for its antioxidant role, 20
and recently has been reported as an important inhibitor of ferroptosis, a non-apoptotic 21
form of cell death. Such death pathways were primarily described in cancer cells, but it 22
has also been identified in hippocampus and renal cells. Here we link the role of this 23
selenoprotein on ferroptosis with possible protective mechanisms of neurodegeneration. 24
Additionally, we propose that selenium (Se) insufficient diet enhance the susceptibility 25
of ferroptosis, as well as other death cell pathways, due to downregulation of GPx4 26
activity. We review recent findings on GPx4 with emphasis on neuronal protection, and 27
associate the relevance of Se on its activity. 28
29
30
Introduction 31
Selenium (Se) is an essential nutrient required to synthesize selenocysteine (Sec), the 32
21st amino acid, which is incorporated into biomolecules by translational coding during 33
selenoprotein synthesis. Twenty-five different selenoproteins have thus far been 34
identified in human proteome1. Among them, the glutathione peroxidase (GPx) family, 35
compound by 8 sequentially numbered isoenzymes that catalyze the reduction of H2O2 36
of organic hydroperoxides by glutathione (GSH) or other biological reductants. 37
Although they are all in the same family, each enzyme has various characteristics that 38
determine their biological role (Table 1). Only GPx1, 2, 3 and 4 are considered 39
selenoproteins in all mammals, as they incorporate Sec as part of catalytic site; GPx6 is 40
considered a selenoprotein in humans though not in rodents; and GPx5, 7 and 8 use 41
cysteine (Cys) in place of Sec2. 42
In the brain, GPx enzymes are expressed in neurons and glial cells3, 4, where their free 43
radicals scavenging role protects against oxidative stress. GPx4 is the most widely 44
expressed isoform in brain, existing as a membrane anchored glycoprotein5 that 45
functions to reduce a wide range of complex hydroperoxy lipids, and also accepts 46
various thiols as reductants6. GPx4 was recently recognized as a key regulatory factor in 47
ferroptosis, a newly discovered non-apoptotic programmed cell death pathway 48
characterized by iron-dependent metabolic dysfunction that causes a rapid elevation in 49
the levels of reactive oxygen species7. Further, the GPx4 is essential for development 50
and cell survival; evidenced in animal models that had Sec replaced by serine in the 51
GPx4 catalytic site8. As the function of the GPx family is key to normal development 52
and cellular metabolism via the regulation of oxidative stress, GPx4 dysfunction is a 53
potential Achilles’ heel for cell survival. 54
In this Perspective, we discuss the function of GPx4 in the brain, and suggest this 55
enzyme may be a key regulator of neurodegeneration. In doing so, we also examine the 56
essential function of GPx4 and the association of Se nutritional status and 57
supplementation, describing the potential benefits on neuronal maintenance via 58
promoting GPx4 activity and expression. 59
60
Biological function of GPx4 61
GPx4, as well as the other Se-containing GPx enzymes, is recognized by its antioxidant 62
role, and has the catalytic center characterized by a tetrad comprising Sec hydrogen-63
bonded to the nitrogen of asparagine (Asn), glutamine (Gln) and tryptophan (Trp) 64
residues9. Four different GPx4 were identified: cytosolic and mitochondrial GPx4, both 65
coded by all 7 exons; sperm nuclear GPx4, which is encoded by an alternative exon in 66
the first intron10, 11; and GPx4-I, which was recently detected in immortalized mouse 67
hippocampal neuron cells (HT22) and is coded by the intron sequence between exons 68
1b and 212. Cytosolic-GPx4 is ubiquitous in cells and the main isoform in neurons. Its 69
activity has been either observed in both membrane and soluble compartments13. 70
Mitochondrial and sperm nuclear GPx4 are predominantly expressed in testes, but also 71
found in neurons. 72
All GPx4 isoforms are distinct from other members of the GPx family, as it exists as a 73
monomer. The ability of GPx4 to reduce complex hydroperoxy phospholipids and 74
cholesterol is partially due to absence of an internal sequence of 20 amino acids forming 75
a surface-exposed loop that regulates substrate specificity in other GPx molecules10, 14. 76
The decreased substrate specificity also supports GPx4 having a wider range of 77
reducing substrates that allow it to function when GSH levels are low. In diseases where 78
high production of reactive oxygen and nitrogen species (ROS/RNS) coincides with low 79
GSH levels, as observed in some psychiatric disorders15 and neurodegenerative 80
diseases16, the low specificity of GPx4 for reducing substrates is likely to contribute to 81
the diverse maintenance roles it has in neurons. 82
GPx4 reaction kinetics share similarities with GPx1 and 3; characterized by three steps 83
following a ‘ping-pong’ mechanism, where bimolecular reactions between the enzymes 84
and substrate sequentially comprise catalytic cycles9. As the reaction mechanism 85
involves the oxidation of Se by hydroperoxide without the formation of any enzyme–86
substrate complex, the enzyme is never completely reduced in vivo, and thus the 87
reaction rate depends on the concentration of GPx4 and hydroperoxides, and not on the 88
concentration of GSH. This implies a relevant difference between the physiological 89
process and the conditions in vitro, because under controlled conditions, hydroperoxide 90
and GSH concentrations are close to equimolar and reactions tend to be more dependent 91
on GSH levels2. Under conditions of hydrogen peroxide signaling, the membrane 92
anchored form of GPx4 would be particularly susceptible to over-oxidation to selenic 93
acid22, thus allowing signaling to occur consistent with the ‘flood-gate’ model of 94
signaling23. 95
Ablation of GPx4 or expression of enzymatically inactive GPx4 is embryonically 96
lethal8, 24, and thus only conditional knockout or heterozygous mice can be studied. 97
Mitochondrial GPx4 null mice are available, although males are infertile due to 98
abnormalities in sperm maturation25, 26. Sperm nuclear GPx4 is required for the 99
structural integrity of mammalian sperm chromatin27, and cytosolic-GPx4 is essential 100
for embryonic survival and development, as may compensate the antioxidant and anti-101
apoptotic role in the absence of the other isoforms. It is suggested that cytosolic-GPx4 102
can also be found in the mitochondrial intermembrane and in the nucleous28, 29. GPx4 is 103
predominantly expressed in developing brain, and neuronal GPx4 null mice are not 104
viable30. Conditional knockout of GPx4 in developed mice at 6 to 9 months of age 105
exhibit hippocampal neurodegeneration, indicating the necessity of GPx4 for brain 106
development and maintenance31. 107
108
GPx4 and oxidative stress in the brain 109
Increased markers of oxidative stress have been identified in Alzheimer’s disease32-34, 110
Parkinson’s disease35, 36, multiple sclerosis37, 38 and amyotrophic lateral sclerosis39, 40. 111
The brain has particular characteristics that result in increased vulnerability to oxidative 112
stress. It has the highest metabolic activity compared to any other tissue, as it requires 113
constant production of large amounts of ATP and resultant byproducts of mitochondrial 114
function to maintain neuronal homeostasis. The brain accounts for only 2% of the total 115
body mass, yet consumes 25% of its energy41. All brain cells produce high levels of 116
nitric oxide (NO) for signal transduction, which amplifies the potential for peroxynitrite 117
formation. Further, neuronal plasma membranes are rich in polyunsaturated fatty acids 118
(PUFAs) that are particularly vulnerable to free radical attack and peroxidation of 119
unsaturated carbon-carbon bonds. 120
In the brain, antioxidant mechanisms exist in a synergy between small molecular weight 121
antioxidants (e.g. ascorbate, and vitamin E) to directly neutralize ROS and RNS; and 122
enzymatic systems comprised of catalase, superoxide dismutase and the glutathione 123
peroxidases. GPx4 is synthesized endogenously in the brain (Figure 1) found 124
predominantly in neurons of the cerebellum, hippocampus and hypothalamus6. 125
However, following brain injury, this selenoprotein is upregulated in reactive astrocytes 126
of damaged areas, indicating protective role counteracting cellular deterioration 127
throughout the brain42. Under conditions of brain injury or neurodegeneration key lipid 128
biomarkers of nitrative and oxidative stress are elevated including the nitrotyrosine, 129
nitro-tocopherol and lipid oxidation products (e.g. malondialdehyde, acrolein and 4-130
hydroxynonenal)43-46. The key role of the lipid oxidation products in ferroptosis and the 131
role of GPx4 in detoxification indicate the crucial role of Sec in GPx4 healthy cell 132
maintenance. 133
GPx4 has anti-apoptotic role due its capacity to inhibit peroxidation of cardiolipin. 134
Because cytochrome-c only binds to cardiolipin (CL), but not to its hydroperoxide state 135
(CL-OOH), the protection of cardiolipin suppress the release of cytochrome-c from 136
mitochondria47 (Figure 2b). GPx4 also modulates ATP generation under oxidative 137
conditions48, which has potential implications regarding mitochondrial dysfunction in 138
Alzheimer’s49 and Parkinson’s diseases50. However, due to the high levels of PUFAs in 139
neurons, peroxidation is perhaps the most relevant oxidative stress process associated 140
with neurodegeneration. Recently, Seiler et al.30 described an apoptotic mechanism 141
induced by lipid peroxidation resulting from 12/15-lipoxy-genase activities, and 142
highlighted the importance of this pathway in neuronal cells. This process results in 143
translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus, 144
leading to large-scale DNA fragmentation and cell death (Figure 2a). In the brain, ROS 145
and RNS are released constantly through neurotransmission and mitochondrial activity. 146
Physiologically, these molecules are either reduced spontaneously in the cytoplasm, or 147
enzymatically processed by superoxide dismutase, resulting in the production of 148
hydrogen peroxide (H2O2), which easily permeates cell membranes if not neutralised by 149
GPx or catalase. Hydrogen peroxide also reacts with redox-active copper or iron via the 150
Fenton/Heiber Weiss reaction, and is converted to a hydroxide anion and hydroxyl 151
radical that favours the formation of lipid peroxides (-LOOH). Lipid peroxidation is a 152
particularly damaging cycle, as these reaction products also act as triggers for the 153
generation of additional lipid peroxides in the membrane via lipoxygenases that catalyze 154
the oxygenation of polyunsaturated fatty acyl groups to hydroperoxides. In this 155
scenario, 12/15-lipoxygenase is relevant because only a low amount of peroxide is 156
needed and it can oxidize complex lipid esters even when incorporated in membranes or 157
lipoproteins51. Interestingly, it has been shown that GPx4 ablation results in propagation 158
of lipoperoxydation cascade via activation of 12/15-lipoxygenase, and treatment with α-159
tocopherol efficiently prevented this apoptotic response activated by GPx4 deficiency. 160
Reactive nitrogen species have a prominent role in the pathology of neurodegenerative 161
diseases. In Alzheimer’s disease, Parkinson’s disease and motor neuron disease 3-162
nitrotyrosine is a biomarker of neurodegeneration, as are protein carbonyls that can 163
result from peroxynitrite-induced oxidation of proteins. The CNS produces 164
approximately 20 times more NO than the cardiovascular system. Nitric oxide is a 165
critical secondary messenger, however in the presence of superoxide it will react at 166
diffusion limited rates to produce peroxynitrite52, making it likely to be a key oxidant in 167
the brain (Figure 2c). Selenium containing compounds have a 250-800 fold faster 168
reactivity with peroxynitrite than corresponding thiol containing compounds53. GPx 169
enzymes and the lipophilic selenium-containing molecule Ebselen (2-Phenyl-1,2-170
benzoselenazol-3-one) are protective against peroxynitrite. Ebselen is a biomimetic of 171
GPx function and has protective effects against lipid peroxidation54, 55. The increased 172
electrophilicity of Sec compared to Cys and the decreased pKa of Sec indicate that 173
GPx4 is a key defense enzyme to protect against peroxynitrite induced lipid 174
peroxidation. Thus, a key function of GPx4 and potentially other selenoenzymes in the 175
CNS could be the protection of the cell from peroxynitrite. In addition to GPx4 being a 176
scavenger of organoperoxides it is also a peroxynitrite reductase56, 57. The reaction of 177
peroxynitrite with GPx has been calculated to be a more efficient substrate for GPx than 178
hydrogen peroxide58. 179
180
Selenium: key element for GPx4 activity in neurodegeneration 181
In vivo studies have shown that Cys can replace Sec in different selenoproteins, as these 182
analogous amino acids differ only in the substitution of selenol moiety by a thiol 183
group59, 60. In the same way, GPx isoenzymes, including GPx4, have Cys homologues61, 184
however the presence of Sec confers increased activity to the Se-containing GPx 185
enzymes59, 60, 62. Indeed, studies designed in an Escherichia coli expression system 186
showed that a recombinant Cys mutant of GPx4 had a significant depletion of catalytic 187
efficiency and 1000-fold lower activity compared to the natural enzyme63-65. This is due 188
to the more acidic pKa of Sec (pKa = 5.5)66-68 compared to Cys (pKa = 8.3). The Sec 189
provides an alternative solution to controlling the pKa of the reactive site residues than 190
modification of the local structure of the protein. Concurrently, the local structure can 191
also influence the pKa of Sec in proteins further69, as occurs in a dramatic shift in the 192
pKa of Cys that can be as low as ~3 for thiol:disulfide interchange proteins DsbA70, 71. 193
Mannes et al.72 showed that, at a physiological levels, Cys-GPx4 prevented death of 194
murine embryonic fibroblast cells in GPx4 knockout mice via an anti-apoptotic 195
mechanism. However, to obtain a comparable function to natural GPx4, more Cys 196
mutant was required. Incorporation of Cys instead of Sec in selenoproteins is dependent 197
of Se availability to the cells60, thus Se deficiency directly impacts on GPx4 production 198
and activity in the brain. Studies have shown the positive effects of Se supplementation 199
in recovering GPx4 activity. Sodium selenite (Na2SeO3) treatment restored GPx1 and 200
GPx4 activity in oxidatively stressed methamphetamine-treated SH-SY5Y cells73. 201
Similarly, mice with induced-neurotoxicity by patulin had increased mRNA levels of 202
GPx1 and 4 after treatment with selenomethionine74. 203
The advantage of Sec compared to Cys is important in the central nervous system, as 204
pH in synaptic vesicles varies constantly. Synaptic transmission causes strong 205
acidification in the synaptic cleft due to release of protons, which is subsequently 206
followed by increase in extrasynaptic pH75. Thus, under acidic pH, Sec would be 207
deprotonated more quickly while thiol would still exist as -SH. Considering that GPx4 208
responds to Se supplementation, we hypothesize that Se supplementation might improve 209
GPx4 activity in different tissues by increasing the Sec to Cys ratio. Indeed, deficient Se 210
status in humans have been associated with risk for Alzheimer’s and Parkinson’s 211
diseases76-79 and the supplementation with a natural source of highly bioavailable 212
selenomethionine improved cognition in mild cognitively impairment patients80. In 213
general, Se status is positively correlated with total GPx activity when measured in the 214
same compartment81. However, some questions arise when total GPx activity is used in 215
order to assess GPx4 function, as: i) there is no known correlation between total 216
peripheral GPx activity and GPx4 in brain, as the isoenzyme GPx3 and GPx1 are the 217
most abundant variants in plasma and erythrocytes, respectively82, 83; ii) circulating 218
GPx4 is low, which makes assessment with adequate sensitivity and specificity 219
difficult; and iii) total plasma GPx activity reaches a plateau when whole blood Se 220
levels reach 1.3 µmol/L84. It is unknown what Se concentration is required for GPx4 221
activity to reach a plateau in the brain, and both in vitro and in vivo studies will help to 222
elucidate the best Se dietary intake strategy that may contribute to increasing GPx4 223
activity in the brain as a means to intervene in neurodegenerative diseases progression. 224
Selenoprotein synthesis is modulated by refined mechanisms that control gene 225
transcription, RNA processing, translation and also post-translational steps of protein 226
biosynthesis. Both selenoprotein synthesis and the hierarchical mechanisms that 227
distribute Se among tissues are tightly regulated, and it is believed that during periods of 228
Se deficiency these mechanisms prioritize synthesis most important selenoproteins and 229
distribution to organs with the highest need85, 86. In vivo studies show that brain, 230
reproductive and endocrine organs have the highest priority for Se uptake and retention 231
during Se deficiency87-89. Although levels of Se in the brain are low (~0.03 µg g-1 wet 232
tissue) compared to other organs, the importance of Se in normal neural function has 233
been demonstrated in studies where competition between high priority organs has been 234
manipulated89. We postulate that dietary insufficiency of Se or impaired transport to the 235
brain contributes to a decreased capacity of neurons to cope with the oxidative and 236
nitrative stress, depleting an individual’s resilience to developing neurodegenerative 237
disease. 238
239
GPx4 and ferroptosis in neurodegeneration 240
Ferroptosis is characterized by metabolic dysfunction that causes increased production 241
of reactive species of oxygen via an iron-dependent mechanism7, 90. In its first step, 242
cysteine/glutamate antiporter system xc− is inhibited, and thus GSH biosynthesis is 243
reduced (Figure 2d). As a consequence, GPx4 activity is negatively affected, resulting 244
in increased lipid peroxidation91, 92. Although lipid peroxidation probably initiates 245
outside the mitochondria independently of 12/15 lipoxygenase, oxidized mitochondrial 246
phospholipids demonstrate effects within this organelle. Proteomic analysis has 247
suggested that GPx4 is the sole member of the GPx family playing a central role as 248
regulator of ferroptosis92. However, it remains unclear if other isoforms have an as-yet 249
undiscovered contribution, and thus additional research on other members of the GPx 250
family is needed to elucidate their involvement in this important new mechanism of 251
programmed cell death. 252
Ferroptosis has been identified in cancerous7, 92 and hippocampal cells7; and it has also 253
been described as a trigger of acute renal failure91. Recently, Chen et al93. reported the 254
participation of this mechanism in neurodegeneration. Adult (3-4 months of age) GPx4 255
neuronal inducible knockout transgenic mice treated with tamoxifen for GPx4 ablation 256
presented a striking paralysis phenotype. Interestingly, only cerebral cortex and 257
hippocampal cells were not sensitive to reduced GPx4 activity, and so it remains 258
unclear why different neuronal cells are disposed to ferroptosis, and if different forms of 259
stress specifically activate ferroptosis in determined cells, such as elevated brain iron. 260
Consistent with the importance of lipid peroxidation driving ferroptosis, alpha-261
tocopherol was protective and we therefore hypothesis that Ebselen would provide 262
protection as well. 263
In light of these data reinforcing the relevance of GPx4 in neuronal health, it is 264
important to better understand the molecular basis of this selenoenzyme in order to 265
optimize its activity as possible strategy for addressing neurodegeneration. Moreover, it 266
is still unclear what effects Se treatment may have in reducing ferroptosis, and future 267
studies should consider the bioavailability of different Se compounds. For instance, it is 268
known that organic Se, as selenomethionine, has high availability and low toxicity, as 269
can non-specifically substitute methionine in serum proteins, especially albumin83. In 270
contrast, inorganic forms as selenite (SeO32-) and selenate (SeO4
2-) have lower 271
bioavailability and higher toxicity (reviewed by Thiry et al.94). Increased understanding 272
of the biochemical role of Se in ferroptosis could provide novel pathways for targeted 273
drug development to treat disease where ferroptosis is a key mechanism. 274
275
Modulating the role of GPx4 as a neuroprotective agent 276
The antioxidant role of GPx4 can be potentiated by association with other biologically 277
active molecules, and this should be considered with regard to strategies designed to 278
minimize neurodegeneration. For instance, N-acetylcysteine (NAC), a Cys-donor and 279
biosynthetic, acts as precursor to GSH and was proven to prevent cell death from 280
eracin-induced ferroptosis in vitro92 (Figure 2d). Other studies have showed NAC has 281
antioxidant activity95, 96, and further experiments using physiological conditions are 282
necessary to demonstrate a potential interaction of NAC with GPx4 in prevention of 283
ferroptosis. 284
Docosahexaenoic acid (DHA) (22:6n-3) is the most abundant n-3 long-chain PUFA in 285
the brain and has indirect antioxidant role associated with regulation of GPX4 gene 286
expression. Hippocampal HT22 cells treated with DHA showed increased expression of 287
GPX4 by around 50% after 48 hours. This regulation appeared to be exclusive to GPX4, 288
as the isoenzyme 1 gene was not affected and no changes in its activity were observed12. 289
On the other hand, a low-DHA diet also led to the stimulation of expression of all GPx4 290
isoforms in wild type animals, which suggests the occurrence of a compensatory genetic 291
strategy to protect cellular membrane from peroxidation under DHA deficiency97. Other 292
mechanisms by which DHA can act as a beneficiary to brain GPx4 activity have been 293
described before, but these data specifically reinforce the associated mechanisms of 294
different antioxidants and presents new avenues for optimizing ferroptosis inhibition as 295
a viable therapeutic strategy. 296
Vitamin E (namely α-tocopherol, the most abundant isoform) is a potent antioxidant and 297
is associated with GPx4 via a chain-breaking electron donor mechanism. In the brain, 298
this micronutrient is at a low concentration, though the radical quenching reaction is 299
extremely fast (~ 108 M−1 s−1)98. Alpha-tocopherol inhibits ferroptosis in vitro7, and 300
GPx4 neuronal inducible knockout transgenic mice treated with a vitamin E enriched 301
diet showed a delayed paralysis phenotype linked to ferroptosis93. However, it is worth 302
mentioning that vitamin E is dependent on the reduction of vitamin C, and so excessive 303
supplementation might have a counterproductive pro-oxidant effect and induce 304
ferroptosis. We hypothesize that under physiological levels, DHA and vitamin E 305
availability to neuronal cells may be important regulators of ferroptosis by influencing 306
GPx4 levels and activity in the brain (Figure 2d), and suggest that the nutritional status 307
of these particular nutrients should be considered when interventions are made in order 308
to optimize GPx4 activity as a strategy to inhibit neurodegeneration. Thus the 309
nutritional status of vitamin E, of which deficiency is widely prevalent99, and Se may be 310
key for optimal health and resilience against oxidatively driven activation of ferroptosis, 311
particularly in neurodegenerative diseases. 312
313
Conclusions 314
Se deficiency has been linked to increased oxidative stress and neurodegenerative 315
diseases. However, different mechanisms may be intrinsic and here we propose that 316
ferroptosis is another path by which Se has an important role in the maintenance of a 317
healthy brain. Selenium is key factor for GPx4 expression and activity, and in deplete 318
situation, selenoproteins present reduced activity due incorporation of Cys instead of 319
Sec, which has negative implications for GPx4 activity and may increase susceptibility 320
of the cell to oxidative stress and induction of ferroptosis. 321
We claim for further studies focused on elucidating the role of Se in both this newly-322
discovered mechanism of cell death, as well the possible association with other small 323
molecules, such as NAC, DHA and α-tocopherol in order to establish new therapeutic 324
strategies to prevent and delay diseases that affect millions of the people worldwide. We 325
believe that optimization of nutritional status of Se may result in higher GPx4 activity 326
and thus delay, or even prevent, neuronal loss. Increasing Se levels is likely to only 327
contribute to a decreased risk in development of neurodegenerative disease in 328
populations that have a decreased Se exposure. Understanding the role of Se proteins, 329
oxidative stress and ferroptosis in neurodegeneration may provide a unique insight to 330
the cellular death mechanisms that occurs in neurodegeneration. 331
332
Figure 1: Mechanism of blood-brain barrier transit of selenoprotein P (SelP) and 333
resultant effects on brain selenoprotein synthesis. Selenium delivery into brain is 334
dependent on selenoprotein P (SelP), which is endocytosed by apolipoprotein E 335
receptor-2 (ApoER2) at the blood-brain barrier and releases Se into the interstitum. 336
Astrocytes then resynthesize SelP to raise a pool of Sec available to the brain as 337
required. ApoER2 is also expressed in neurons, and is the likely neuronal import route 338
for SelP. GPx4 is synthesized endogenously in neurons, obtaining the necessary Se 339
from SelP transit across the neuronal membrane via the ApoER2, which increases the 340
intracellular Sec:Cys ratio and stimulates transcription of a range of selenoproteins, 341
including GPx4100. 342
343
Figure 2: Glutathione peroxidase 4 modulates different pathways to inhibit neuronal 344
loss. 2a. GPx4 reduces activation of 12/15-lipoxy-genase, inhibiting the translocation of 345
AIF from the mitochondria to the nucleus, which leads to large-scale DNA 346
fragmentation and cell death; 2b. In mitochondria, GPx4 inhibit the peroxidation of 347
cardiolipin (CL) and then suppress the release of cytochrome-c from mitochondria and 348
apoptosis signalling; 2c. GPx4 acts as scavenger of organoperoxide and peroxynitrite; 349
2d. Ferroptosis is characterized by the inhibition of the xc− system, responsible for Cys 350
import, causing limited GSH biosynthesis. As GPx4 can reduce lipid peroxides when 351
GSH levels are low, it is a negative regulator of this cell death pathway. Alpha-352
tocopherol, in a chain-breaking electron donor mechanism, plays antioxidant role in 353
association with vitamin C, and thus is also considered negative regulator of ferroptosis. 354
NAC is a GSH precursor because donates Cys. DHA upregulates GPx4 expression. 355
Abbreviations: AIF: apoptosis-inducing factor; CL: cardiolipin; CL-OOH: cardiolipin 356
hydroperoxide; Cys: cysteine; DHA: Docosahexaenoic acid; GPx4: glutathione 357
peroxidase 4; GSH: glutathione; GSSH: glutathione disulfide; SOD1: superoxide 358
dismutase 1; H2O2: hydrogen peroxide; H2O: water; LOO.: lipid peroxide; NAC: N-359
acetylcysteine; NO: nitric oxide; NO2-: nitrogen dioxide; .O2
-: superoxide; ONOO-: 360
peroxynitrite; RNS: reactive nitrogen species; ROS: reactive oxygen species. 361
362
Acknowledgments 363
We thank the Science without Borders (Ciência sem Fronteiras) Fellowship that 364
supported BRC research. The authors declare no competing financial interests. 365
This work was supported by the Australian Research Council linkage project 366
(Linkage project 140100095); the Victorian Government Operational 367
Infrastructure Support Program; Cooperative Research Centre for Mental Health 368
Florey Neuroproteomics Facility. The authors declare no competing financial 369
interests. 370
371
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Table 1: Characteristics of mammalian glutathione peroxidases.
GPx
type
Peroxidatic
residue
Quaternary
structure
Molecular weight
(kDa)
Reducing
substrate
Subcelullar
location
Principal location
GPx1 Sec tetramer 88.4 (isoform 1)
10.3 (isoform 2)
GSH
Cytoplasm,
cytoson,
mitochondrion
Kidneys, liver,
erythrocytes
GPx2 Sec tetramer 87.9 GSH Cytoplasm Gastrintestinal
mucosa
GPx3 Sec tetramer 102.2 GSH,
thioredoxin,
glutaredoxin
Extracellular Plasma, kidneys,
intestinal villus,
adipose tissue,
extracellular body
fluids
GPx4 Sec monomer 19.5 (cytosolic)
22.2 (mitochondrial)
GSH,
cysteine,
protein thiols
Cytoplasm,
mitochondrion,
nuleus
Testes,
spermatozoa,
brain
GPx5 Cys tetramer 100.8 (isoform 1)
45.7 (isoform 2)
NA Extracellular,
plasma,
membrane
Epididymis,
spermatozoa
GPx6 Sec in humans
Cys in mice
tetramer 99.9 (humans) GSH Secreted Olfactory
ephitelium
GPx7 Cys monomer 21.9 GSH, protein
disulfide
isomerase
Secreted
-
GPx8 Cys - - GSH Cytoplasm -
Cys: cysteine; GPx: glutathione peroxidase; GSH: glutathione; Sec: selenocysteine1 2 3, 4
5.
